Hydrolysis of beta lactam linkage by penicillinase



Patented June 24, 1952 UNITED STATES HYDROLYSIS F BETA LACTAM LINKAGE BYPENICILLINASE Henry Welch, Silver Spring, Md., assignor to the UnitedStates of America No Drawing. Application September 20, 1949, Serial No.116,849

1 Claim. '(01. 195-30) (Granted under the act of March 3, 1883, as

amended April 30,

This invention relates to an enzymatic substance and more particularlyto an enzymatic substance from a species of actinomyces now identifiedas Actinomyces candidus, which acts as an inhibitor or antagonist ofpenicillin, an antibiotic agent.

In the sterility control of penicillin and penicillin-containinsubstances it has been standard practice to mix a solution containingthe penicillin with solutions of antagonists, such as cysteine-H01, orHydroxylamine-HCI, allowing them to remain in contact sufficient timebefore inoculation into various media to insure destruction of theantibiotic activity of the penicillin. In certain instances ataka-diastase preparation commercially available as Cla-rase is used forthe inactivation of penicillin. The penicillin inhibiting materials inClarase are derived from bacterial contaminants of the subtilis-mesen:tericus group during the manufacture of the taka-diastase and are in noWay associated with the metabolic products of the mold itself,Aspergillus cryzae. The penicillin inactivating materials produced inthe preparation of clarase vary widely in the amount present and incertain lots, depending upon the other organisms present, may beentirely absent.

When penicillin has been inactivated by any of the above procedures theinactive material is distributed into portions of culture media andobserved for a period of time for signs of growth. Sensitive organisms,which may be capable of resisting the action of penicillin while in thesporulating stage, may thus be grown out. This is also true ofpenicillin-resistant organisms which may remain dormant in thepenicillin preparations but become viable in the body. Thus thepreparation to be described has decided value in the sterility controlof this antibiotic and its various preparations.

During the therapeutic application of penicillin preparations, it attimes becomes necessary to determine whether the infecting organisms hasbeen eliminated from the patient. The organism may have been inhibitedby the penicillin but not destroyed. In this condition it may remainvirulent and produce a relapse as soon as penicillin therapy iswithdrawn. Blood must be obtained from the patient to determine whetherthe infectious agent has been eliminated. This is difllcult at timesduring the therapy since there is penicillin in the blood stream, oftenin concentrations high enough to inhibit the infecting organisms in theblood withdrawn. The enzyme to be described here rapidly destroys thisresidual blood penicillin when added to the culture flasks and allowsviable organisms present to grow out. Thus the 2 substance is of valuein diagnostic procedures. In the chemical assay of penicillin one methodemploys the specific reactivity of an enzyme preparation specific forthe beta-lactam linkage 5 which exists in penicillin. This enzyme actsby forming a new carboxyl group which is subject to measurement, themolar concentration afi'ording an extremely accurate method of assayingthe amount of penicillin present. When the enzyme to be described ispurified by alcohol and acetone precipitations to concentrate the enzymeand remove interfering buffer systems, it may be employed directly inthis type assay. The assay results obtained agree very closely with allknown chemical methods for assay of penicillin. The assay of crystallinepenicillin G by this method, for instance, gives a value within 4% 0fthe theoretical as a maximum deviation.

In the identity tests for antibiotic agents it often becomes necessaryto use a specific enzyme to differentiate two closely relatedsubstances. The penicillinase to be described is specific for thebeta-lactam structure which in antibiotics occurs only in thepenicillins. Thus this enzyme serves as a reliable method for theidentification of penicillin.

The substances now in use for accomplishing the above have severalinherent disadvantages. Cysteine and hydroxylamine are toxic to manybacteria unless used with certain qualifications which detract fromtheir value. Clarase,.the commercial taka-diastase is not bacteriostatlcbut the production of the penicillin inactivating materials is haphazarddepending upon contamination with poorly defined bacterial agents.

us a desirable preparation of penicillinase is one that can be readilyproduced from an identifiable micro-organism and one which can beproduced consistently and in a yield which will permit practicalapplication.

Briefly restated the penicillinase to be described-concerns an enzymaticsubstance which specifically reacts with penicillins and in so doingafi'ords a means for determining sterility, eflicacy of therapeuticapplication, identity and potency of penicillin andpenicillin-containing preparations. Accordingly, an object of thisinvention is to provide a novel penicillinase in such form that itpossesses practical value. This substance is obtained as a metabolicproduct of growth of an air borne micro-organism which was isolated inthe following manner:

Petri dishes were prepared with sterile nutrient agar containingsufiicient penicillin added just before hardening to give a finalconcentration of 1000 units .per milliliter. 'Thesez plates were thenexposed in various places like dusty attics, on ledges of buildings, instoreroomsietc;

After thirty minutes to two hours the plates were covered and allowed toincubate. Any colonies that grew were removed and subjected to furtherstudy. A typical experiment is detailed in Table 1.

It was assumed that organism of this nature were capable of eitherdestroying the penicillin in its immediate area or of growing inrelatively high concentrations. Bacteria, many of which may producepenicillinase, were not included in this study because past-experienceshowed technical difilculties, particularly filtration problems,preclude large scale production techniques. Only fungi were studiedfurther.

The next step involved determination of whether the fungi actuallydestroyed the penicillin or merely grew in its presence. Liquid culturemedia containing 1000 units of penicillin per milliliter were preparedand inoculated with mycelial transplants of the fungi. These wereincubated at room temperature which is optimum for most fungi, and whengrowth appeared the culture media was assayed to determine amount ofpenicillin remaining. This assay was repeated on the second and thirdday to determine rate of destruction. The experiment, using the fungidescribed in Table 1, is described in Table 2.

All six of the fungi, it should be noted, destroy penicillin to someextent. The outstanding inactivator is M-102, identified as actinomycesvcandidus. An agar slant culture of this particular strain of actinomyceshas been deposited in a permanent collection in the Northern RegionalResearch Laboratories at Peoria, Illinois, under the number NRRL 2280.This is the fungus employed and is the one with which this patentapplication is concerned. In accordance with this invention thisparticular strain of actinomyces is used in production of the penicillinaccording to the following procedure:

Any good nutrient broth is prepared and sterilized in large containersof fermentation tanks. A source of sterile air is supplied in such amanner that it continuously bubbles through the fermentation mixture.The media is inoculated with the fungi, the air supply adjusted andgrowth allowed to proceed at room temperature for five days or until themedium attains a pH of 8.0-8.1, whichever occurs first. The resultingso- 4 lution may be sterilized by passing through a bacterla retainingglass filter or it may be dried and retained in the dehydrated statefrom which it is reconstituted before use.

This preparation may be concentrated by saturating the fermentationmedium after removal of the mycelia with ammonium sulfate and collectionof the precipitate in a small volume of water from which it isprecipitated by the addition of two volumes of alcohol or acetone. Thisprecipitate is then resuspended in water and filtered through glass tosterilize or dehydrated and held in the dry state. This purificationstep is necessary in preparing the penicillinase for use inthe-enzymatic assay of penicillin, since the buffering effect must be ata minimum to allow accurate measurement of the fall in pH.

As illustration of the possible uses for this novel penicillinase aseries of experiments follow.

Table 3 presents data secured using the filtered fermentation mediumfrom a run made exactly as described above.

Table 3 Inactivation in Completc.

Do. Nearly complete. Partial.

The consistency with which this penicillinase was produced wasdetermined by examining five consecutive production batches. These dataare set forth in Table 4.

Table 4 Final Units Pen- Inactiva' pH 1c in tion Hours 8.0 35,000Complete 8.0 35,000 1 Do. 8.1 35,000 1 D0. 8.0 35,000 1 D0. 8.2 35,000 1Do.

This crude material which consisted of the penicillinase, in thepresence of other metabolic products and remnants of culture media constituents, was tested for stability. At 37 C. deterioration was rapid,but at room and at refrigerator temperatures the material was quitestable.

When the crude fermentation broth was dried and reconstituted as a 10%aqueous solution and filtered, it was employed in the routine method fordetermining the sterility of penicillin preparations with the followingresults. (Table 5.)

Table 5 2 Size Penicillin Product mg};

1:. 100,000 Amorpb. Sod. Penicillin Complete 200,000 Amorph. Sod.Penicillin Do. l00,000 Cryst. Sod. Penicillin G Do. $0,000 Cryst Sod.Penicillin G Do. 500,000 Amorp Do. 500,000 Cry Do. 500,000 Cryst. Pot.Penicillin G Do. 1,000,000 Amorph. Calcium Peniciliin Do.

9 1,000,000 Amorph. Sod. Penicillin Do.

10 l,000,(ll0 Cryst. Bod. Penicillin G Do.

ll 1,000,000 Cryst. Pot. Penicillin G Do.

When this 10% suspension was used in the inactivation of penicillin inoil and wax, a somewhat more difiicult procedure, the following resultswere obtained.

Table 6 Amount of Penicillinasc Used Sam- Couple mining I 0.05 ml. 0.1ml. I 0.2 ml. j 0.51111.

i units 1 l 30,000 2 so, 000 l I Inactivated.

When the penicillinase was used in blood cultures, inactivation ofpenicillin present was complete. When employed in the purified state forthe assay of penicillin the results were identical with those obtainedby the sodium hydroxideiodometric titration method.

The invention described herein may be manufactured and used anywhere byor for the Government of the United States for governmental purposeswithout the payment to me of any royalty thereon in accordance with theprovisions of the act of April 30, 1928 (ch. 460, 45 Stat. L. 467).

REFERENCES CITED The following references are of record in the file ofthis patent:

Florey et al., Antibiotics, Oxford University Press, 1949, p. 1085,1086. 1093, 1095, 1110.

Chem. Abst. 41, 5917(f). A. griseus secretes penicillinase.

Woodrufi' et al., Jour. Bact 47 (1944), pp. 425-6.

McQuarrie et al., Arch Biochem, v. 5, 1944, pp. 307-315.

Chandler et al.. Science 102, Oct. 5, 1945, pp.

